The long-term goals of this project are to synthesize and study nonpolar, non-hydrogen-bonding nucleosides and nucleotides as biophysical probes of noncovalent bonding in DNA-DNA, protein-nucleotide, and protein-DNA interactions. These molecules are designed to mimiC as closely as possible the shape and size of the natural nucleosides, but without polar hydrogen bonding N, N-H and C=O groups. Preliminary studies of several such analogs over the past 1.5 years have shown that they display a number of unusual properties both alone and in the context of DNA helices, and they have already led to a number of surprising and valuable findings in the study of stacking and pairing in duplex DNA and in the study of the fidelity of Klenow fragment of E. Coli DNA Polymerase I. The stacking results are likely to be of practical use in the design of helix-stabilizing agents for oligonucleotide-based therapeutics, and the polymerase studies will aid in the design of novel polymerase inhibitors (with potential antiviral or antineoplastic effects). In the long term, it is hoped that these new analogs will be of general utility in biophysical studies of noncovalent bonding. The proposed studies are aimed at the use of these novel analogs in three separate biomolecular systems, both as a test of the properties and utility of the compounds, and also to shed new light on noncovalent interactions in these systems. Experiments are planned for the study of base stacking in DNA, noncovalent bonding requirements in nucleotide incorporation by DNA polymerases, and energetic contributions to DNA binding by TATA-binding proteins. Specific plans for the term covered by this proposal include: (1) ln-depth study of the six current analogs (and their natural counterparts) as probes of thermodynamic factors of base stacking in DNA; (2) Synthesis of several new analogs to gain additional data in stacking studies and in polymerase experiments; (3) In-depth study of the processing of these analogs by E. Coli Pol I and three other polymerases; (4) Use of these analogs to test energetics of specific interactions between yeast TATA-binding protein and a consensus TATA recognition sequence.